Retrotransposition-independent LINE-1-induced DNA damage in normal and aging cells PROJECT SUMMARY Genomic structural variants (SVs), including insertions and deletions (indels), make a significant contribution to human diversity and disease. Numerous causes for indels and SVs have been proposed, including replication slippage, recombination, unequal crossing over, and imperfect repair of DNA double- stranded breaks. This is a proof of principle proposal to show experimentally that LINE-1 (L1) retrotransposons may also significantly stimulate genomic instability, such as that accompanying aging, to a degree that exceeds canonical retrotransposition insertions. Retrotransposons are mobile DNA elements that duplicate themselves by a copy and paste mechanism using an RNA intermediate. L1s comprise at least 17% of human DNA. While it is thought about 100 remain competent for retrotransposition in any human individual, many more are transcribed. L1 retrotransposition has also been responsible for the insertion of over a million non-autonomous Alu retrotransposons and thousands of processed pseudogenes. L1s pose an ongoing threat to the human genome. The second open reading frame (ORF2) of the L1 encodes endonuclease activity capable of making double-stranded breaks in DNA. It is reasonable to suggest that some proportion of these breaks are repaired by the cell's non-homologous end-joining (NHEJ) DNA repair pathway which is prone to generation of errors and deletions. Thus, elevated expression of L1s and their encoded endonuclease activity, such as occurs during aging, cancer progression, embryogenesis, and in some parts of the brain may increase cellular DNA damage. Using next generation sequencing, we will test this hypothesis in cultured cells and transgenic mice. In Aim 1, the genomes of a transformed cell line and early passage and senescing primary human fibroblasts will be examined for increased DNA damage following transfection of an active L1 construct. In Aim 2, tissues from progeny of young and aged mice will be examined for genomic mutations not marked by an L1 insertion that are generated following the induction of a tetracycline-responsive L1 transgene. If supported, the hypothesis has the potential to improve our understanding of genetic change that accompanies not only aging, but also cancer and various disease conditions associated with failure of DNA repair.